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Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH 1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We veri®ed that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC pro®le to that identical with oxidized CRH standard. In addition, IVB cells exhibited high af®nity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) imunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.Corticotropin-releasing hormone (CRH) is a 41 amino acid containing neuropeptide which coordinates the neuroendocrine, behavioural, autonomic and immune responses to stress (1, 2). It is secreted from the hypothalamus and acts on pituitary corticotrophs to enhance secretion of adrenocorticotropic hormone (ACTH) and other pro-opiomelanocortin products. ACTH, in turn, stimulates glucocorticoid synthesis and secretion in the adrenal cortex. CRH containing parvocellular neurones coexpress vasopressin (VP) and are subject to negative feedback by glucocorticoids following stressful conditions (3).The cellular effects of CRH are mediated by high-af®nity receptors (4±7). In addition to the anterior and intermediate lobes of the pituitary (8), CRH receptors are located in a wide variety of locations in the central nervous system (CNS), including the parvocellular neurones of the paraventricular nucleus where they are induced by stress (9). There are two CRH receptor types.

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